FUNCTIONAL INTERROGATION OF RIBOSOMAL BIOLOGY USING CONTINUOUS EVOLUTION PROJECT SUMMARY/ABSTRACT: In nature, fundamental biological phenomena that are central to cellular life are inherently hindered from probing and interrogation, as these dynamic systems cannot be easily decoupled from immediate artifactual disruptions throughout the living cell. One such case is the ribosome, a colossal multi-component protein factory that functions as the nexus for cellular information and signaling events, integrating nutrient availability with growth dynamics and resource allocation. Despite decades of research, this biomolecular assembly remains superficially understood and underexplored, owing to the difficulty associated with decoupling the translational apparatus from cellular viability. In fact, there is currently no generalizable experimental tool-kit for unbiased high-throughput interrogation of the structure-activity and functional relationships of the ribosome, the prediction of ribosome-small molecule interactions, or the identification of disruptive resistance mechanisms. The work proposed herein seeks to overcome the challenges associated with ribosomal manipulation in vivo, to illuminate the relationship between the rRNA and the effective translation initiation/elongation rates as they relate to growth fitness, and to provide an innovative framework for the interrogation of ribosome-small molecule interactions. The proposed work focuses on the development of a fully orthogonal ribosomal system for the real-time monitoring of ribosome activity in living cells through engineered transcription-translation networks based on independently tunable genetic components at all stages. The designed orthogonal sensor ribosomes will be subjected to directed evolution yielding novel variants with enhanced or diminished kinetic properties. To achieve this, the orthogonal ribosome circuit will be interfaced with an emergent technique based on a continuous culturing methodology called Phage-Assisted Continuous Evolution (PACE), facilitating hundreds of rounds of directed evolution in just a few days with minimal researcher intervention. Finally, to demonstrate the utility of the newly developed ribosomal sensors and the evolutionary platform, this technology will be leveraged to inform antibiotic-ribosome interactions, and to generate actionable drug resistance profiles for delaying or evading microbial resistance. This platform will be extended to high-throughput screening campaigns for novel chemical scaffolds capable of modulating ribosomal translation through potentially undiscovered modes of action. Broadly, our ability to harness bacteria for biomedical and biomaterial applications in the future will hinge on the detailed understanding of the mechanistic control and optimization of ribosomal output parameters enabled by these studies. The technological advances proposed herein have the potential to extend our understanding of key factors governing ribosomal function and dynamics, and will pave the way towards the development of novel mechanisms that will illuminate and enhance new approaches in biomedical research and targeted antimicrobial therapeutics.